Enzyme activity that can liberate phosphoric acid from certain precursor compounds has been known since the beginning of this century. These enzymes, known as phosphatases, have a ubiquitous distribution in biological systems, although relatively little is known about their biological function.
Recently, phosphatases have been used as detectable labels in a number of analytical assays based on the use of enzymes as labels for a member of a specific binding pair. These assays rely on the recognition between members of the binding pair and the detection of a label on one member of the binding pair to indicate the presence of the other member. Examples of binding pair members include antibodies (including binding fragments) and antigens, nucleic acid probes (both DNA and RNA) and their conjugates, carbohydrates and lectins, and hormones and their receptor proteins.
Use of a phosphatase in either a histological staining process or as a detectable label requires a substrate for the phosphatase that undergoes a detectable change as a result of the action of the enzyme on the substrate. The general types of substrates known to exist for phosphatases include esters and amides of phosphoric acid. Of these, mono- and di-esters of alcohols and phenols have been utilized for the most part. In general, the nature of the organic radical does not affect the specificity of the reaction. Phosphatases also hydrolize the S-P bond of thioesters. A number of phosphatase substrate types are set forth in the following table.
TABLE ______________________________________ Phosphatase Substrate Types* ______________________________________ 1. Alk--O*P Phosphate esters of alkyl alcohols (Alk = alkyl grouping) 2. Ar--O*P Aryl phosphate esters (Ar = aryl grouping) 3. Ar--Alk--O*P Arylalkyl phosphate esters 4. C = C--O*P Enol phosphates (e.g., phosphopyru- vic acid) 5. R--CO--O*P Acyl phosphates (e.g., acetyl phosphate) 6. (ArO).sub.2 *P Diaryl phosphates 7. P*OP Inorganic pyrophosphate 8. (Ar,Alk)P*OP Organic pyrophosphate 9. Ar.sup. --N*P Phosphamide (Ar.sup. = aryl grouping, creatine, or arginine) 10. (Ar,Alk)S*P Thioesters ______________________________________ *Location of bond being cleaved.
Examples of specific techniques used in dyeing of tissue samples include the metal-salt phosphatase technique that depends in its original version upon the enzymatic release of phosphate ions that are precipitated as insoluble calcium phosphate. The calcium phosphate is then visualized, for example by conversion to cobalt sulfide, conversion to silver phosphate followed by exposure to light, reaction with either sodium alizarin sulfonate or phthalocyanine dyes to form a calcium lake, or reaction with pentahydroxyflavone to form a fluorescent lake.
Another dyeing technique that also releases a visible component for use with enzyme labels in analytical techniques is the azo-dye technique. This technique relies upon the coupling of various components (under either alkaline or acid conditions) after enzymatic hydrolysis of phosphate esters of the coupling components. The released coupling agent couples with a diazonium component to form a highly colored azo dye. In a modification of this technique, known as a postcoupling procedure, incubation with enzyme is carried out in the absence of a diazonium salt, which is added later. General configuration of typical phosphate esters used in the azo-dye method are shown in the formulas below: ##STR1## In these formulas, (A) through (F) indicate the location of various substituents, which can be either electropositive and/or electronegative. The asterisk indicates the point of hydrolysis upon action of phosphase, and the arrow indicates the coupling position in the naphthol to the diazonium salt component (the broken arrow indicates a less reactive coupling position). Commonly used diazonium salts are shown in the following formulas, in which (+) represents an electropositive substituent, (-) represents an electronegative substituent, and (A) through (J) indicate the position of various groupings in known compounds that can be either (+) or (-). ##STR2##
An additional technique of visualization is based on the oxidation of indoxyl substrates, such as 3-indoxyl phosphate, to indigo dyes. The principle of the indoxyl technique is shown in the reaction scheme set forth below. ##STR3##
A fourth technique is the indoxyl phosphate/tetrazolium salt method in which at an alkaline pH the production of indigo decreases and the production of colorless dehydroindigo increases. The hydrogen released by the formation of indigo or dehydroindigo reduces colorless soluble tetrazolium salt to colored insoluble diformazan at the enzyme site as shown in the following exemplary scheme. ##STR4##
There are a number of problems associated with available phosphatase substrates used in the methods set forth above. For example, the most widely used diazonium salts in the azo-dye method are arylamine diazonium chlorides and analogous simple salts. These compounds are unstable in solution and require refrigeration in dry form for storage. Even at low temperature, slow decomposition occurs over a period of time. Commercial techniques of stabilization include the use of aluminum, magnesium, and zinc sulfates; magnesium oxide; magnesium bicarbonate; and disodium naphthalene-1,6-disulfonate. Even though small quantities of diazonium salts are used, the metallic and other stabilizers exert a marked inhibitory affect upon phosphatases and other enzymes that may be present (such as oxidases). Additionally, diffusion artifacts occur when there is time for soluble components released from an enzyme reaction to diffuse away from the reaction site before precipitation and/or color formation occurs. Accordingly, there remains a need for improved phosphatase substrates that are stable without the inhibiting stabilizers used with various substrates now available and which will be insoluble and highly colored immediately upon release without requiring the occurrence of other reactions after the phosphatase reaction is completed.